Monoclonal antibodies against the extracellular domain of human VEGF-receptor protein (KDR)

ABSTRACT

Monoclonal antibodies, secreted by hybridoma cell lines, that are directed against an epitope of the extracellular domain of human VEGF-receptor KDR, methods of determining human VEGF-receptor KDR in cell lysates or tissue analysates and the use of the antibodies in analytical assays, in diagnostics and as carrier molecules for therapeutic substances, are described.

The present invention relates to monoclonal antibodies, secreted byhybridoma cell lines, that are directed against an epitope of theextracellular domain of human VEGF-receptor KDR (kinase insert domaincontaining receptor), to methods of determining human VEGF-receptor KDRin cell lysates or tissue analysates and to the use of the antibodies inanalytical assays, in diagnostics and as carrier molecules fortherapeutic substances.

New vessels form as capillaries which sprout from existing smallvessels. This process, known as angiogenesis, takes place in response tocertain signals (Alberts et al., Molekularbiologie der Zelle, VerlagChemie, Weinheim).

Angiogenesis is a process that is meticulously controlled by theinteraction of vascular endothelial growth factor (vascular endothelialgrowth factor) and its corresponding highly affine KDR-receptor onendothelial cells. KDR has been characterised as a transmembranaltyrosine kinase receptor of sub-type 5 which serves as a key regulatorof vascular endothelial cell development during embryogenesis and cellregeneration (Cancer and Metastasis Reviews 15: 159-163, 1996).Furthermore, the dysfunction of that normally meticulously regulatedligand-receptor interaction results in impairment of the angiogenesisprocess, which is a feature of many illnesses. Particularly important isthat the growth of tumours and their metastases has proved to beangiogenesis-dependent to a high degree.

A monoclonal antibody having a limited reaction pattern is alreadyknown. That antibody is directed against the extracellular domain ofmouse KDR-homologous flk-1 and is capable of neutralising the VEGFstimulation of a chimeric flk-1/fms-receptor that is expressed intransfected 3T3 cells (Rockwell et al., Molecular and CellularDifferentiation, 3 (1): 91-109 (1995)).

Monoclonal antibodies that can be used for a broad spectrum ofanalytical assays and methods of determining human VEGF-receptor KDR incell lysates and tissue analysates using such antibodies have not beenknown hitherto.

The aim of the present invention is to provide monoclonal antibodiessecreted by hybridoma cell lines. The monoclonal antibodies according tothe invention can be used for a broad spectrum of analytical proceduresand assays, in diagnostics and as carrier molecules for therapeuticsubstances.

There have now been prepared monoclonal antibodies that are directedagainst an epitope located within extracellular domains 1 to 7 of humanVEGF-receptor KDR.

The present invention relates especially to monoclonal antibodies thatare directed against an epitope located within extracellular domains 6and 7 of human VEGF-receptor KDR.

The monoclonal antibodies AM 2-7-9, AM 2-10-1, AM 5-1-2 AM 5-10-13 andAM 2-4-1 are preferred.

The monoclonal antibodies AM 2-7-9 and AM 2-10-1 are especiallypreferred.

Those antibodies are directed specifically against an epitope that islocated within extracellular domains 6 and 7 of human VEGF-receptor KDR.

The antibodies according to the invention exhibit a high degree ofselectivity in a broad spectrum of analytical procedures and assays.

The monoclonal antibodies according to the invention have the advantagethat they neither interfere with the ligand-binding domain nor activateKDR after binding.

The antibodies according to the invention are also valuable fordiagnostic purposes.

For example, the monoclonal antibodies according to the invention can beused in Western blots, immunoprecipitation, ELISA, FACS analysis and inindirect immunofluoresence microscopy.

A further use of the monoclonal antibodies is in immunohistochemistry.

The monoclonal antibodies can also be used in screening for smallagonistic and antagonistic molecules and in the detection of mutantreceptor subtypes.

The monoclonal antibodies according to the invention can also be used indiagnostics, it being possible to couple them in combination with asuitable contrast-enhancing substance. The antibodies react with anepitope that is not located within the ligand-binding side of the KDRand is therefore capable of reacting with all KDR-receptor populations.

A preferred contrast-enhancing substance that can be coupled to theantibodies is technetium-99.

The monoclonal antibodies according to the invention can also be used inangiogenesis-dependent phenotypes, such as tumours and metastasesthereof, rheumatoid arthritis or psoriasis, or pathological symptomsresulting therefrom, when coupled with suitable toxic substances.

Suitable toxic substances are adequately known to the person skilled inthe art and are described, for example, in Byers & Baldwin, Immunology(1988), 65, 329-335 and in Blakey et al., Waldmann H. (ed): MoncclonalAntibody Therapy, Prog. Allergy. Basle, Karger, (1988), vol. 45, 50-90.

The invention relates also to the use of mRNA that codes for the heavyand the light chain of the antibodies according to the invention in thepreparation of recombinant “single-chain antibodies”.

The invention relates also to the use of the recombinant antibodiesencoded by mRNA for Western blots, immunoprecipitation, ELISA and FACSanalysis, in indirect immunofluoresence microscopy andimmunohistochemistry and in screening for small agonistic andantagonistic molecules and in the detection of mutant receptor subtypes.

The invention relates also to a method of determining humanVEGF-receptor KDR in cell lysates or tissue analysates, characterised inthat

1. the monoclonal antibodies AM 2-7-9, 2-10-1, AM 5-1-2, AM 5-10-13 orAM 2-4-1, as captor antibodies, are coupled in purified form at aconcentration of 1-10 μg/ml in coupling buffer on ELISA plates and thenexcess binding sites are blocked with blocking buffer,

2. cell or tissue analysates are prepared in a suitable lysis buffer,

3. the plates are washed in washing buffer before application of thelysates,

4. the KDR-protein to be determined in the sample is quantified using arecombinant KDR-protein as standard curve,

5. the tissue and cell lysates to be analysed are then introduced intothe test system,

6. incubation is carried out at room temperature for 2 hours,

7. the ELISA plates are then washed.

8. determination of the “captured” KDR is carried out by means of apolyclonal anti-KDR antiserum and finally

9. detection by means of a chromogenic, chemiluminescent or radioactivesubstance is carried out.

The method can also take place in the form of a kinase test procedurethat determines tyrosine-phosphorylated KDR.

In order to allow quantification of the test method, recombinantphosphorylated KDR can be used as the standard protein.

The determination of human VEGF-receptor KDR in cell lysates or tissueanalysates can be carried out both qualitatively and quantitativelyusing the method.

The polyclonal anti-KDR antiserum used in step 8 of the method can be,for example, polyclonal anti-phosphotyrosine antiserum or a monoclonalanti-phosphotyrosine antibody.

The detection carried out in step 9 of the method can preferably becarried out with peroxidase-labelled secondary antibodies and suitablechromogenic or chemiluminescent substrates.

The antiserum used can be, for example, from goats or rabbits or fromsheep, rats or donkeys, either directly in the form of enzyme-labelledimmunoglobulin or indirectly by means of enzyme-labelled anti-goat,anti-rabbit, anti-sheep, anti-rat or anti-donkey antibodies. Eitheralkaline phosphatase or peroxidase is used for that purpose.

The ELISA plates used in step 1 of the method can be antibody-coated andblocked.

The lysis buffer used in step 2 of the method contains 1-5 mM divalentions and 1-15% glycerol.

The lysis buffer preferably contains 1-2 mM divalent ions and 5-12%glycerol.

A lysis buffer that contains 1.5 mM divalent ions and 10% glycerol isespecially preferred.

A preferred divalent ion is, for example, magnesium.

The washing and dilution buffers used in the method can be any buffersknown to the person skilled in the art for that purpose.

Preferred washing buffers are those containing PBS with 0.05% detergentand 0.1% bovine serum albumin.

Preferred dilution buffers are those containing 1% bovine serum albumin.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a gel with the specific reaction pattern using the exampleof the monoclonal antibody AM 2-10-1:

track 1: pig aorta endothelial cells, 25 μg total cell lysate

track 2: pig aorta endothelial cells, transfected with human KDR, 25 μgtotal cell lysate

track 3: CHO cells, transfected with human KDR, 25 μg total cell lysate

track 4: CHO cells, 25 μg total cell lysate

track 5: human umbilical cord endothelial cells, passage 6, 25 μg totalcell lysate

track 6: mouse endothelial cells, 25 μg total cell lysate

FIG. 2 shows the gel for immunoprecipitation with the monoclonalantibodies AM 2-10-1 and AM 2-7-9:

track 1: 5 μg AM 2-7-9

track 2: 1 μg AM 2-7-9

track 3: 5 μg AM 2-10-1

track 4: 1 μg AM 2-10-1

track 5: isotype control, 10 μg non-specific antibody of subclass IgG1

FIG. 3 shows epitope mapping:

track 1: soluble KDR, domains 1 and 2

track 2: soluble KDR, domains 1 to 3

track 3: soluble KDR, domains 1 to 5

track 4: soluble KDR, domains 1 to 7

The following Examples describe the preparation of monoclonal antibodiesaccording to the invention and use thereof, but the invention is notlimited to these Examples.

1. Preparation of Monoclonal Antibodies

Female Balb/c mice (six weeks old) are hyper-immunised either withsoluble receptor (20 μg/injection) in Freund's complete adjuvant or withbacculovirus-infected SF-9 cells (10⁷ cells/injection in PBS) thatexpress the full-length human KDR receptor protein. Specific antibodytitres are determined using the extracellular KDR domain by ELISA. Threedays after the last immunisation the spleen is removed. The splenocytesare fused with SP20.Ag.14 murine myeloma cells in accordance with knownmethods (Köhler and Millstein, Nature 256, 495, 1975). The coloniesformed are screened by means of ELISA and Western blot using the solubleextracellular KDR domain. Positive colonies are cloned three times bylimited dilution, purified with protein A/Sepharose chromatography andfurther characterised.

2. Use in Western Blots

Complete cell lysates are prepared by lysis in a suitable lysis bufferthat contains the above-described concentration of divalent ions andglycerol. Equal amounts of protein are separated by means ofpolyacrylamide gel electrophoresis under nonreducing conditions,transferred to nitrocellulose and probed with the monoclonal antibodiesAM 2-7-9 and AM 2-10-1 in accordance with procedures known per se. Thevisualisation of the immunoreactive bands is carried out either by meansof chemiluminescence or enzymatically, usingalkaline-phosphatase-labelled secondary antibodies and correspondingchromogenic substrates. FIG. 1 shows the gel of a Western blot.

3. Use in Immunoprecipitation

Immunoprecipitations are carried out using a suitable lysis buffer (50mM Tris-HCl pH 7.4, 150 mM NaCl, 1% IGEPAL® CA-630, 10% (v/v) glycerol,1.5 mM MgCl₂, 1 mM EGTA, 5 mM EDTA, 50 mM NaF, 2 mM sodiumortho-vanadate) and complete protease inhibitors in accordance withknown methods. FIG. 2 shows the gel of the immunoprecipitation.

4. Use in Immunohistochemistry

Cells are cultured on sterile cover glasses until shortly beforeconfluence and are fixed with formaldehyde (4% v/v PBS per 4 g per litreglucose) before or after incubation with 20 μg/ml of the correspondingantibody in PBS. The immunoreaction is visualised withalkaline-phosphatase-labelled secondary antibodies and fast naphthol redas chromogenic substrate.

5. Epitope Mapping

2 μg portions of recombinant soluble KDR-receptor domains that areprovided with a “myc-tag” are separated by SDS-PAGE (10%) andtransferred to nitrocellulose. Then two identical films are incubatedwith the monoclonal antibody AM 2-10-1 or with a monoclonal anti-mycantibody. Immunocomplexes are visualised byalkaline-phosphatase-labelled secondary antibodies (see FIG. 3).

What is claimed is:
 1. A monoclonal antibody that is directed against anepitope located within extracellular domains 1-7 of human VEGF-receptorKDR and is not reactive with the ligand binding site of theVEGF-receptor KDR.
 2. A monoclonal antibody according to claim 1,wherein said antibody is directed against an epitope located withinextracellular domain 6 or extracellular domain 7 of human VEGF-receptorKDR.
 3. A method for identifying epitopes located within theextracellular domains 1-7 of the VEGF-receptor KDR comprising reactingthe monoclonal antibody according to claim 1 with a sample containing aVEGF-receptor KDR in a Western Blot assay, a immunoprecipitation assay,an ELISA assay, a FACS assay or an indirect immunofluorescence assay. 4.A method for identifying epitopes located within the extracellulardomains 1-7 of the VEGF-receptor KDR comprising reacting the monoclonalantibody according to claim 1 with cells expressing the VEGF-receptorKDR in an immunohistochemistry assay.
 5. A method for screening smallagonistic and antagonistic molecules in the detection of mutant receptorsubtypes comprising assaying cells for the mutant receptor by reactingsaid cells with the monoclonal antibody according to claim
 1. 6. Amethod for diagnosing cells bearing VEGF-receptor KDR comprisingreacting said cells with the monoclonal antibody according to claim 1wherein the method is an immunological method.
 7. The method accordingto claim 6, wherein the monoclonal antibody is coupled to a suitablesubstrate contrast-enhancing substance.
 8. The method according to claim7, wherein the contrast-enhancing substance coupled to the antibody istechnetium-99.
 9. A method of determining human VEGF-receptor KDR in acell lysate or tissue analysate comprising: (a) coupling a monoclonalantibody which is directed against an epitope located within theextracellular domains 1-7 of human VEGF-receptor KDR, but is notreactive with the ligand binding site of the VEGF-receptor KDR, inpurified form at a concentration of 1-10 μg/ml in coupling buffer, to anELISA plate and then blocking excess binding sites with a blockingbuffer, (b) preparing a cell or tissue analysate in a suitable lysisbuffer, (c) washing the ELISA plate in washing buffer before applicationof the analysate, (d) introducing the cell or tissue analysate of step(b) to be analysed into said ELISA plate to form a test system, (e)incubating the test system to allow VEGF-receptor KDR to bind saidmonoclonal antibody and thus to capture said VEGF-receptor KDR, (f)washing the ELISA plate to remove unbound material, and (g) detectingthe captured VEGF-receptor KDR using a secondary antibody in achromogenic, chemiluminescent or radioactive assay system.
 10. Themethod according to claim 9, wherein the VEGF-receptor KDR can beassayed using a kinase assay.
 11. The method according to claim 9,wherein recombinant phosphorylated KDR is used as the standard protein.12. The method according to claim 9, wherein the second antibody is ananti-phosphotyrosine antibody.
 13. The method according to claim 12,wherein the anti-phosphotyrosine antibody is monoclonal.
 14. The methodaccording to claim 9, wherein the substance used for detection in step iof the method is a peroxidase-labeled secondary antibody which is thendetected by using a suitable chromogenic or chemiluminescent substrates.15. The method according to claim 9, wherein the lysis buffer used instep b of the method contains from 1 to 5 mM divalent ions and from 1 to15% glycerol.
 16. The method according to claim 15, wherein the divalention is magnesium.
 17. The method according to claim 9, wherein the lysisbuffer used in step b of the method contains from 1 to 2 mM divalentions and from 5 to 12% glycerol.
 18. The method according to claim 9,wherein the lysis buffer used in step b of the method contains 1.5 mMdivalent ions and 10% glycerol.